Mammalian cells are an established expression system in the biotechnology industry for the production of recombinant proteins (r-proteins). In contrast to lower eukaryotes or prokaryotes, mammalian cells provide active r-proteins that possess relevant post-translational modifications. However, in order to obtain sufficient amount of protein for structure/activity analyses or high-throughput screenings, one needs to go through the long and tedious process of stable clone isolation and characterization. Protein production by large-scale transfection is an interesting alternative to the generation of stable clones as it allows the very fast generation of mg to gram quantities of r-protein within few days.
The use of vectors containing the Epstein-Barr virus (EBV) oriP in cell lines stably expressing EBV's EBNA1 protein, such as the HEK293-EBNA1 (293 E) cell line (ATCC#CRL-10852) significantly increases protein yield (Durocher et al., 2002). EBNA1 is a multi-functional protein that have been shown to positively regulate many viral promoters present on plasmid DNA when the oriP is present in cis (Reisman and Sugden, 1986).
The production of secreted r-protein often needs to be performed in serum-free medium in order to facilitate their purification. Adaptation of the 293E cell line to serum-free medium formulations is not straightforward and is rarely successful. To circumvent this problem, the generation of new 293-EBNA1 cell line from a serum-free medium adapted 293 cell line is preferable (Pham et al., 2003; Pham et al., 2005). However, these new cell lines do not always show optimal growth properties or high transfectabilities in serum-free medium. Also, the isolation of new clones stably expressing full-length EBNA1 is difficult as this protein seems to be cytotoxic to the cells.
Preliminary transient gene expression studies with the commercially available 293F cells adapted to the FreeStyle™ medium showed that this cell line has a good potential for the large-scale r-protein production in serum-free medium. Improvement of this cell line by stably expressing a less cytotoxic but functional EBNA1 protein is needed.
Kennedy, G. and Sugden, B. (2003) EBNA-1, a Bifunctional Transcriptional Activator Molecular and Cellular Biology, 23: 6901-6908 disclose that the ability of EBNA1 to activate transcription from both integrated and transfected templates can be inhibited by a derivative of EBNA1 lacking the amino acids required for activation from integrated templates (aa 65-89). We have found, against previous expectations, that truncations of these amino acids from EBNA1-coding nucleotide sequences can enhance transient gene expression in HEK293 cells to a level similar to EBNA1.